Epidemiological studies show that HDL-cholesterol (HDL-C) level correlates inversely with risk of cardiovascular events. The most prominent atheroprotective action of HDL particles involves the transport of excess cholesterol from peripheral tissues to the liver for excretion into bile and then feces, a process known as reverse cholesterol transport (RCT). Recently, there has been increased interest in raising the HDL-C level of individuals, but established drug therapies that substantially increase HDL-C are scarce and their efficacy controversial. One approach is to enhance the functional efficacy of RCT and other pathways involved in cholesterol removal. Scavenger receptor class B, type I (SR-BI), which mediates 'selective' uptake of cholesteryl esters from HDL (or other lipoprotein) particles, is highly expressed in liver and steroidogenic tissues. In liver, SR-BI-mediated selective uptake of CE from HDL potentiates RCT by increasing hepatic excretion of cholesterol. SR-BI also helps to clear excess cholesterol by mediating selective delivery of HDL-CE to steroidogenic cells of the adrenal glands and gonads for storage or steroid production. We recently reported two observations. First, we demonstrated that two insect orthologs of SR-BI-fruitfly CG3829 and silkworm Cameo2-are much more efficient at mediating selective uptake of HDL-CE in in vitro transfected cells as well as in vivo in the liver of mice overexpressing CG3829 (LivCG3829Tg) as compared with mammalian (rat) SR-BI. Second, deficiency of NHERF1 or NHERF2 leads to enhanced SR-BI function in hepatic and steroidogenic cells of the adrenal glands and gonads. The experiments outlined in this proposal will utilize novel insect constructs encoding SR-BI orthologs, designer cells, genetic knockout animal models, and state-of-the-art molecular, cellular, biochemical, and physiological techniques to provide important insights into the beneficial actions of SR-BI having enhanced functional efficiency for removing excess cholesterol. Aim 1 will characterize the functional significance of insect orthologs of SR-BI in liver, adrenal, and gonadal tissues in vivo and in vitro. Aim 2 will examine the impact of deficiency of NHERF1 and/or NHERF2 on SR-BI expression, selective CE transport, and RCT in liver in vivo and in vitro. Aim 3 will examine the impact of deficiency of NHERF1 and/or NHERF2 on SR-BI expression, selective HDL-CE uptake, and steroid production in the adrenal glands and gonads in vivo and in vitro. We expect that these studies will provide new insights into the mechanism by which increased SR-BI efficiency influences RCT, biliary cholesterol secretion, cholesterol removal by steroidogenic tissues via its catabolism bile acids and steroids. Such understanding will potentiate the development of new therapies to treat cardiovascular disease and other diseases related to dysfunction of lipid metabolism, bile acids, and steroids and fatty liver disease.